Project Summary/Abstract As a key risk factor for vascular and neurodegenerative disease, aged American individuals 65 years and older are expected to compose ~25% of the United States population by 2030. In particular, 95% of the patients with Alzheimer?s Disease (AD) and related dementias are contained within this aged population, whereby the cost of care is projected to increase from the current (2018) $280 billion to an unsustainable $1.1 trillion in 2050. With key mechanisms yet to be fully understood, scientific evidence has consistently shown that cardio- and cerebrovascular health regulated by endothelial cell (EC) function is integral to maintenance of a healthy brain. Cerebrovascular ECs coordinate dilation of blood vessel networks in the brain to regulate blood flow in a manner that meets the metabolic demand of neurons. Thus, the goal of the proposed research is to resolve the role of EC function in cerebral blood flow regulation during cerebrovascular aging and AD. Parallel vasodilator signaling pathways underlying EC function include the production of nitric oxide (NO) and activation of small- and intermediate-conductance Ca2+-activated K+ (SKCa/IKCa) channels. While age-related endothelial dysfunction has been defined as a reduction in NO signaling, effects of aging and AD on SKCa/IKCa channel signaling remain unclear. Further, mitochondrial production of hydrogen peroxide (H2O2) may coincide with the aging process and development of AD. We will test the central hypothesis that cerebrovascular endothelial SKCa/IKCa channel activity is dysregulated, resulting in a corresponding impairment of cerebral blood flow during oxidative stress with advancing age and AD. To comprehensively investigate the physiological & pathological coupling of mitochondrial Ca2+ homeostasis and activation of SKCa/IKCa channels, our laboratory employs novel examination of blood flow and cerebral perfusion, intact posterior cerebral arteries, and cerebral endothelial tubes of NIA C57BL/6, mitochondrial catalase overexpressing (or mCAT), and AD (3xTgAD) mice (age range: 3 to 30 months, male & female). Aim 1 seeks to determine functional relationships between intracellular Ca2+ organelles (mitochondria, endoplasmic reticulum), Ca2+-permeant transient receptor potential (or TRP) channels, and SKCa/IKCa channels in isolated endothelium. Aim 2 addresses structural alterations in connectivity among ECs and smooth muscle cells coupled with the contribution of endothelial SKCa/IKCa function to vasoreactivity. Finally, in Aim 3, we will develop therapeutic strategies for improving cerebrovascular aging and AD using optimized endothelial-dependent regulation of cerebral blood flow. As a novel avenue for genetic and pharmacological treatment, we will pursue fine-tuning of signaling inputs that govern SKCa/IKCa channel activity in the cerebral vasculature spanning from molecular approaches to the whole organism; reconciling scientific mechanism with therapy. In this manner, the proposed Aims identify possible therapeutic strategies that promote effective blood flow regulation during conditions of advancing age and AD while optimizing vascular signaling to sustain the quality of life in older humans.